Method for production of {60 -galactosidase

ABSTRACT

In culturing an Alpha -galactosidase-producing mold in a known medium, addition of organic acids such as citric acid, lactic acid and glycolic acid to the medium strikingly enhances the Alpha -galactosidase activity of the enzyme to be consequently produced. When the enzyme is used for the purpose of hydrolyzing the raffinose contained in the beet molasses-including syrup or juice in the process of beet sugar production, the enhanced Alpha -galactosidase activity serves to decrease the amount of enzyme required and facilitate the enzyme handling.

United States Patent Watanabe et al.

[ METHOD FOR PRODUCTION OF a-GALACTOSIDASE [75] Inventors: Masayuki Watanabe; Shigeyoshi Narita; ltiro Kagaya; Minoru Miura; Yasunobu Suzuki; Shuji Tanaka; Masami Sasaki; Shogo Miyatani; Kenichi Terayama, all of Kitami, Japan [73] Assignee: Hokkaido Sugar (10., Ltd., Tokyo,

Japan [22] Filed: Jan. 28, 1974 [21] Appl. No.: 437,535

[52] U.S. Cl 195/65; 195/114 [51] Int. Cl Cl2d 13/10 [58] Field of Search 195/65, 66 R, 114, 11

[56] References Cited 7 UNITED STATES PATENTS 3,795,585 3/1974 Suzuki et al. 195/65 [451 July 15, 1975 Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firrril(urt Kelman [57] ABSTRACT In culturing an a-galac tosidase-producing mold in a known medium, addition of organic acids such as citric acid, lactic acid and glycolic acid to the medium strikingly enhances the a-galactosidase activity of the enzyme to be consequently produced. When the enzyme is used for the purpose of hydrolyzing the raffinose contained in the beet molasses-including syrup or juice in the process of beet sugar production, the enhanced a-galactosidase activity serves to decrease the amount of enzyme required and facilitate the enzyme handling.

3 Claims, N0 Drawings METHOD FOR PRODUCTION OF a-GALACTOSIDASE BACKGROUND OF THE INVENTION This'invention relates to a method for the production of a-galactosidase by the culture of a mold. More particularly, the present invention relates to a method for the production of a-galactosidase by the culture of a mold, the a-galactosidase activity of said enzyme being strikingly enhanced by adding certain types of organic acids to the medium in which the culture is carried out. a-Galactosidase has heretofore been known as an enzyme capable of hydrolyzing raffinose into sucrose and galactose and it isextensively utilized in beet sugar production. In addition,it is widely used'as a reagent for the determination of raffmose contents and as a reagent for the determination of chemical structures of compounds When the a-galactosidase is used for the purposes mentioned above, the enzyme is desired to have strong d galactosidase activity but to be weak in the invertase activity which is simultaneously manifested. As a mold productive of an enzyme combining strong a-galactosidase activity with weak invertase activity, there has heretofore been known Mortierella vinacea var. raffinoseutilizer (ATCC 20034) (US. Pat. No. 3,647,625). Other molds which have a similar behavior include type strains belonging to the genus of Absidia such as, for example, Absidia lichtheimi (Lucet et Costantin) Lendner (IFO 4009), Absidia lichtheimi (Lucet et Costantin) Lendner (IFO 4010), and Absidia reflex a van Tieghem (IFO 5874), type strains belonging to the genus of Circinella such as, for example, Circinella muscae (Sorokine) Berlese et de Toni (Circi'nella sydowi Lendner) (IFO 4457) and Circinella mucoroides Saito (Circinella Chinensis Naganishi et Kojiro) (IFO 4453), and Circinella muscae (Berlese et de Toni) nova typica coreanus (ATCC 20394) which has rec ently been discovered by one of the present inventors.

SUMMARY OF THE INVENTION It is an object of this invention to provide, in the production of a-galactosidase by the culture of a mold, a method for strikingly enhancing the a-galactosidase activity of the enzyme to be produced within the mycelia of said mold.

To accomplish the object described above, the method according to the present invention causes a medium used for culturing an a-galactosidaseproducing mold to incorporate therein 02 to 1.0 percent, based on the medium, of organic acids such as citric acid, lactic acid and glycolic acid. Thea-galactosidase activity of the enzyme to be consequently produced can be strikingly enhanced by thus adding said organic acids, as promoters, to the known medium. For the use to which this enzyme is put, the enhanced a-galactosidase activity proves highly advantageous since it leads to decreasing the amount of enzyme required and also to facilitating the enzyme handling in the reaction involved.

Other objects and characteristics of the present invention will become apparent from the detailed description of the invention and the preferred embodiments to be given herein below.

DETAILED DESCRIPTION OF THE INVENTION various avenues of research after a possible measure for enhancing the a-galactosidase activity of the enzymes to be produced within the mycelia of said molds. Consequently, they have discovered that the a-galactosidase activity of the enzymes is strikingly enhanced and the invertase activity thereof is hardly increased by adding to the known medium such organic acids as citric acid, lactic acid and glycolic acid.

Although the method of this invention can be applied to all the a-galactosidase-producingmolds without exception, it is particularly effective on molds of the genus Absidia and the genus Circinella. As type strains of the genus Absidia on which the present invention is effective, there can be cited Absidia lichtheimi.(Lucet et Costantin) Lendner (IFO 4009), Absidia reflexa van Tieghem (IFO 5874), Absidia hyalospora (Saito) Lend- .ner (IFO 8082), Absidia ramosa (Vuillemin) Lendner (IFO 8083) and Absidia regnieri (Lucet et Costantin) Lendner (IFO 8084), for example. Examples of type strains of the genus Circinella on which this invention is effective include Circinella muscae (Sorokine) Berlese et de Toni (C. W. Hesseltine) (IFO 6410), Circinella muscae (Sorokine) Berlese et de Toni (Circinella lydowi Lendner) (IFO 4457), Circinella mucoroides Saito (Circinella chinensis Naganishi et Kojiro) (IFO 4453) and Circinella mucoroides Saito (K. Saito) (IFO 4455). Further, Circinella muscae (Berlese et de Toni) nova typica coreanus (ATCC 20394), a newly discovered mold deposited at ATCC on Jan. 7, 1974 can likewise be used. This novel mold has been isolated from Korean yeast cake by one of the present inventors. Although the attributes of this mold are well in arrangement with those of Circinella muscae (Sorokine) Berlese et de Toni described in the report of C. W. Hesseltine and Dorothy I. Fennel (The genus Circinella,

Mycologia Vol. XLVII, No. 2, 193-212, March-April,

1955), a difference is recognized in terms of appearance and color tone of the microorganic flora in the initial stage of culture. In addition, these two molds clearly differ in physiological properties, particularly, a-galactosidase producing ability. As a new type of the species Circinella muscae, the mold of the invention has been designated Circinella muscae (Berlese et'de Toni) nova typica coreanus, The mycological characteristics of this mold are as follows: i

A microscopic observation reveals that 'the sporangiophores grown on the Synthetic Mucor Agar medium assume. a light brown color and produce sympodial branches as they get old. Their forwardends invariably form circinate coils downwardly, with a single sporangium borne at the extremity of each coil. Occasionally, sterile spines branch off near sporangia and they are separated by septa. Main sporangiophores and branched sporangiaphores are slightly different in size from each other, the former measuring 8 to 10 microns and the latter 4 to 6 microns respectively in diameter. Sporangia have a spherical form or a slightly pressed pseudospherical form and measure30 to 65 microns in diameter. At first, they are white and gradually assume a dark gray color as they mature. They are observed to vary in diameter; small ones measure about 20 microns and large ones are found to reach as much as microns. The wall surfaces of sporangia are slightly coarse and broken and frequently retain dished collars in the lower portion of columella.

Columella measure 10 to 30 by 30 to 40 microns on the average and occur in various forms such as oval,

conical, pear-shaped and rectangular form. Some have a smooth Surface and others have an indefinite number of protuberances at the top. They assume a light gray color. I l

Sporangiosphores have a spherical to pseudospherical form with a smooth surface and'rneasure .4 to 6 microns in diameter. They appear practically colorless when they are observed individually. When in the form of a cluster, however, they appear to have a dark gray to black color. Growth of chlamidospores or zygosphores is observed.

Macroscopic observation reveals that the mycelium grown on synthetic mucor agar medium, potato dertrose agar medium and malt extract agar medium at first presents a felt-like appearance which changes in color from white to dark gray or black as thesporangia mature. The top of old cultures is covered by light brown sporangiophores having a dirty appearance. This is caused by growth of the light brown sporangiophores which reach to 7 mm. There is no production of soluble pigment in the medium.

The medium to be used for the method of this invention is prepared by combining carbon sources, nitrogen sources and inorganic salts which are usually employed in culturing molds and further incorporating therein such inducers as lactose, galactose, raffinose and melibiose.

As carbon sources usable for this purpose, there can be cited starch, glucose, glycerine, maltose, dextrine, sucrose and invert molasses. Examples of nitrogen sources include soybean flour, peanut powder, ground cottonseed, corn steep liquor, meat extract, peptone, yeast extract, nitrates and ammonium salts. Inorganic salts usable for the medium include common salt, potassium chloride, magnesium sulfate, manganese sulfate, iron sulfate, phosphates and calcium carbonate, for example. If occasion demands, vitamins may be incorporated.

According to the method of this invention, organic acids are added as growth promoters for a-galactosidase to the basal medium which is prepared of the foregoing essential components. Desirably, the amount of such organic acids to be added falls in a range. of be tween 0.2 and 1.0 percent based on thebasal medium. The enhancement of the a-galactosidase activity which the present invention aims to achieve in the produced enzyme is not obtained when the amount of added organic acids falls outside'the range mentioned above; As organic acids which are usable for incorporation to the basal medium, there may be cited citric acid, lactic acid, glycolic acid, fumaric acid, glutaric acid, malic acid, galacturonic acid, tartaric acid, succinic acid and pyruvic acid. In consideration of economy and enhancement of a-galactosidase activity, however, citric acid, lactic acid and glycolic acid prove to be most practical for the purpose.

The mold inoculated to the medium incorporating such promoting substances can be aerobically cultured by an ordinary method in its unmodified form. To be specific, the mold is inoculated to a medium prepared by adding a prescribed amount of a-galactosidase growth promoters to a basal medium formed of suitable carbon sources, nitrogen sources, inorganic salts and inducers and subjecting it to shaken culture or aerated culture at about 30C for 40 to 72 hours, with the pH value maintained in a range between 5 and 8. The culture liquid consequently obtained is separated by a known method into a liquid portion and a mycelial mass. After the mycelial mass is washed with water, it can be added in its unaltered form to beet molasses so that the enzyme contained therein may act on and hydrolyze the ratfinose contained in the beet molasses. The mycelial mass which has been washed with water may be treated by a suitable known method so as to be put to a desired application.

As is clear from the foregoing explanation, the method of the present invention can increase the total amount of a-galactosidase produced by the mold by merely adding organic acids to the medium conventionally employed for culturing molds. Since the produced enzyme shows highly enhanced a-galactosidase activity per weight of enzyme and substantially no increase in invertase activity, it has the economic advantage that the concentration adjustment of the beet molassesto be treated canbe accomplished easily and the amount of enzyme required for the treatment can be decreased.

Now, the preferred embodiments of the present invention-and comparative examples will be cited herein below. It should be understood, however, that the present inventionis not limited to the preferred embodiments in anyway.

EXAMPLE I A basal medium was prepared by using 1.0 percent of lactose, 1-0 percent of glucose, 1.0 percent of corn steep liquor, 0.6 percent of (Nl-l SO 0.3 percent of KH PO 0.2 percent of MgSO .7l-l O and 0.2 percent of NaCl. The basal medium was divided into nine portions and one of the organic acids listed in Table l was dissolved in each portion in an amount of 0.7 percent. Then, the medium of each portion was adjusted to pH 5.0 and 0.3 percent of CaCO ,was..-adde d thereto. To ZOO-ml of. each portion of the medium-. -th prepared, Absidia hyalaspora (Saito) Lendner (IFOSOSZ) in the form of spore suspension was inoculated in an amount to give a count of l X 10 spores per cc .of medium and shaken cultured at 30C for 48 hours,.-,with the agitation performed at a rate of 136 r.p.m. The results were as shown in Table l.

By way of comparison, the culture was performed by following the procedure described above, except for addition of organic acids. The results are indicated in Table l. a

Referring to the date shown in table, the values of a-galactosidase activity are those determined by adding 1 ml of mycelia suspension under testto a mixture of 0.5 ml of 0.06M melibiose and 0.5 ml Q fOJM phosphate buffer solution (pH 5.2) to permit reaction to ensue at 40C for 2 hours, thereafter heating the reaction mixture in a boiling water bath for 5 minutes to inactivate the enzyme, then adding 1 ml of 1.8% Ba- (OH) .8H O and 1 ml of 2% ZnSO .7H O to the reaction solution to deprive the solution of protein, centrifuging the resultant mixture and assaying the resultant protein-free supernatant for glucose content by the glucostat process. In consideration of the fact that the amount of glucose liberated from melibiose and the enzyme concentration are in a proportional relationship up to 1,000 pg of glucose, the suspension was diluted sugar content by the Somogyi-Nelso'n process. The in- 10 vertase activity which produced 1 pg of invert sugar under the conditions mentioned above was taken as 1 unit. v h

The dry mycelial weight was found by drying at 105C that amount of mycelia which has been grown in 100 ml of culture medium and'thereafter" weighing the dried mass of mycelia.

EXAMPLE 2. V

A basal medium was prepared by using 1.0 percent 5 of lactose, 1.0 percentof glucose, 0.3 percent of (NH SO 0.5 percent of yeast extract, 0.5 percent of peptone, 0.2 percent of K HPO 0.05 percent of MgSO .7H O, 0.002 percent of MnSO .4-6H O, 0.001 percent of 1 50 711 0. The basal medium was divided into 10 portions and one of the organic acids listed in Table 2 .was dissolved in. each portion in an amount of 0.7 percent. Then, the medium of each portion was adjusted to pH 5.8 and 0.3 percent of CaCO was added "thereto; To ZOO-m1 of each portion of the medium. Circinella muscae (Berlese et de Toni) nova typica corcanus (ATCC 20394) .in the form of spore suspension was inoculated in an amount to give a count of 1 X 10 Table 1 Residual Dry weight a-Galactosidase lnvertase Organic acid sucrose (g) of activity activity as glucose mycelium Total I Spec. act. Total Spec. (g/100 ml) in 100 ml act. (I /g on act. act. L/g

(l /m1) dry basis) (a/ml) on dry basis) Glycolic acid 0.05 1.36 185,600 "1,365 10' 92 6,796 Citric acid 0.05 1.40 172,900 "1,235 10" 95 6,806 Lactic acid 0.09 1.34 168,400 1,257X10" 91 6,825 Succinic acid I 0.18 1.30 108,600 835 10 85 6,527 Maleie acid 0.03 1.37 103,800 758 10' 89, 6,509 Fumaric acid 0.06 1.35 92,900 688X10" 88 6,555 Galacturonic 0.07 1.37 86,900 634x10" 89 6.480 acid Malic acid 0.02 1.32 82,300 623 10 85 6,430 Tartaric acid 0.02 1.33 80,100 602 10 86 6,445

Comparative Example (no 0.07 1.37 64,500 471Xl 88 6,427 addition of organic acid) It is clear from the foregoing table that when the culture was made in the presence of glycolic acid, citric acid or lactic acid, the a-galactosidase activity increased by about three times and the invertaseactivity remained substantially unincreased as compared with the culture performed in the absence ofsuch organic acid.

spores per cc of medium and shaken cultured at 30C for 48 hours, with theagitation performed at a rate of 136 rpm. The results were as shown in Table 2.

By way of comparison, the culture was performed by following the procedure described above, except for addition of organic acids. The results are indicated in 7 Table 2. i

, Table 2 Residual Dry weight d-Galactosidase lnvertase Organic acid sucrose (g) of activity activity as glucose mycelium Total Spec. act. Total Spec. (g/100 ml) in 100 m1 act. (p/g on act. act. (,uJg

(pijml) dry basis) t/ml) on dry I basis) Citric acid 0.03 1.36 404,300 2,973 10" 64 4,695 Lactic acid 0.03 1.44 389,300 2,703 10 68 4,702 Glycolic acid 0.03 1.50 385,600 2,571 10' 72 4,811 Fumar c ac d 0.03 1.24 325,700 2,627 10" 57 4,600 Glutanc :clCld 0.03 1.51 321,600 2,130X10" 73 4,850 Mallc acid 0.03 1.25 306,000 2,448 10 61 4,905 Galacturonic 0.03 1.33 295,400 2,221X10 62 4,670 acid 1 Tart'ari c acid "0.03 "1.42 275,600 1,941Xl0 63 4,450 Succlnic acid 0.03 1.28 256,500 2,004X' 63 4,920 Pyruvtc alcld Q-Q3, 1.48 231.100 1x10 73 4,920

Comparative Example (no 0.03 1.45 175,000 l,207 10" 65 4,502 addition of i organic acid) EXAMPLE 3 3. By way of comparison, the culture was performed in 10 the absence of organic acids. The results are indicate in Table 3.

8. ganic acid added was in .the neighborhood of 0.7 percent and that said activity, declined when the amount increased or decreased from said level.

1 EXA PLE 4' The media'wer'eprepared by repeating the procedure of Example 2, except that the amounts (0.7 percent) of citric acid, lactic acid and glycolic acid shown in Table 2 were changed respectively to "0.2 percent, 0.4 per-' cent, 0.6 percent, 0.8 percent and 1.0 percent. To the media, Circinella muscae (Berlese et de Toni) nova typ- Table 3 Dry a-Galactosidase lnvertase Organic Amount R.S. weight activity activity acid added as glucose (g) of Total Spec. act. Total Spec.

(%) g/100 mycelium act. (u/g on act. act. (,u/g

ml in 100 ml (,u/ml) dry basis) (IL/m1) on dry basis) 0.3 0.03 1.30 1 15,900 892x10 90 6,923 Glycolic 0.5 0.02 1.34 160,800 1,200X10 91 6,791 acid 0.7 0.04 1.36 175,600 1,291X10 92 6,765

0.3 0.03 1.38 140,200 1.016X10 91 6,594 Citric 0.5 0.03 1.38 156,300 1,133X10" 94 6,812 acid 0.7 0.04 1.41 171,000 1,213 10 94 6,667

0.3 0.02 1.35 164,500 l,219 10 88 6,519 Lactic 0.5 0.02 1.36 183,800 1,351 10 91 6,691 I acid 0.7 0.03 1.36 188,400 1,385X10" 92 6,765

Comparative I Example (no addition of 0.02 1.34 65,300 487X10 89 6,642

organic acid) I It is clear from the foregoing table that the a-galacto- 40 ica coreanus (ATCC 20394) was ,inocu1ated and culsidase activity was highest when the amount of an ortured. The results were as shown in Table 4.

Table 4 Dry a-Galactosidase lnvertase Organic Amount R.S. weight activity activity acid added as glucose (g) of Total Spec. act. Total Spec.

(%) g/lOO mycelium act. (IL/g on act. act. (pig ml in 100 m1 (p/ml) dry basis) (p/ml) on dry basis) 0.2 0.04 1.42 268,100 1,888X10" 63 4,437 Citric 0.4 0.03 1.41 320,900 2,276X10" 63 4,468 acid 0.6 0.03 1.45 407,100 2,8011X10 66 4,552 0.8 0.04 1.47 384,200 2,614 10 66 4,490 1.0 0.03 1.40 345,400 2,467X10" 64 4,571 0.2 0.03 1.43 242,800 1,698X10 64 4,476 Lactic 0.4 0.03 1.45 31 1,300 2,147Xl0" 66 4,552 acid 0.6 0.03 1.52 405,400 2,667X10 68 4,474 0.8 0.03 1.50 353,500 2,357 10 67 4,467 1.0 0.03 1.41 300,000 2,128X10 64 4,5 39 0.2 0.04 1.43 248,200 1,736X10 68 4,755 Glycolic 0.4 0.04 1.46 341,300 2,338X10 72 4,932 acid 0.6 0.03 1.45 397,600 2,742 10 74 5,103 0.8 0.03 1.46 366,200 2,508X10" 73 5,000 1.0 0.04 1.44 319,800 2,221X10 70 4,1161

Comparative Example (M) 0.03 1.41 176,200 1,250 10 63 4,468 addition of organic acid) EXAMPLE 5 To the media prepared by the procedure of E ;;ampl e 2, various type strains of the genus of Circinella and the genus of Absidia were inoculated and cultured under the culture of a mold, belonging to the genus of Cireine11a or Absidia which comprises adding to a basal medium about 0.2 percent to about 1.0 percent, based on said basal medium, of an organic acid selected from the same conditions as described in Example 2. The re- 5 the group consisting of citric acid, lactic acid, glycolic sults are shown in Table 5 (in the rows indicated by the acid, fumaric acid, glutaric acid, malic acid, galactumark). By way of comparison, the culture was perronic acid, tartaric acid, succinic acid and pyruvic acid, formed in the absence of citric acid. The results are and culturing the mold in said prepared medium under likewise shown in Table (in the rows indicated by the conditions enabling the mold to produce a-galactosi- 11 mark). dase.

Table 5 Citric Dry a-Galactosidase lnvertase acid weight activity activity used (0) (g) of Total Spec. act. Total Spec. act. or not mycelium act. (IL/g on act. t/g on used (x) in 100 ml (pt/m1) dry basis) (u/ml) dry basis) Circinella muscae (Sorokine) x 1.36 43,200 318x10 120 8,823 Berlese et de Toni (C.W. Hesseltine) o 1.38 154,000 1,1 16x10 123 8,913 (IFO 6410) Circinella muscae (Sorokine) x 1.40 71,000 507x10 94 6,714 Berlese et de Toni (Circinella sydowi Lendner) (lFO 4457) o 1.41 173,500 1,230 10 98 6,950 Circinella mucoroides (Saito) x 1.37 109,500 799x10 92 6.715 (Circinella chinensis Naganishi et 0 1.37 232,000 1.693X10 99 7,226 Kojiro) (IFO 4453) Circinella mucoroides Saito (K. Saito) x 1.35 102,400 759x10 94 6,963 (IFO 4455) o 1.38 221,600 1,606X10 99 7,174 Absidia reflexa van Tieghem x 1.42 72,500 51 1 10 109 7,676 (IFO 5874) o 1.43 140,500 983x10 119 8,322 Absidia regnieri (Lucet et x 1.35 70,000 519x10 108 8,000 Costantin) Lendner (IFO 8084) 0 1.37 161,700 1,180X10 120 8,759 Absidia lichtheimi (Lucet et x 1.40 101,500 725x10 121 8,643 Costantin Lendner (lFO 4010) 0 1.42 208,600 1,469 10 150 10,563 Absidia lichtheimi (Lucet et x 1.40 93,600 669x10 120 8,571 Costantin) Lendner (IFO 4009) o 1.41 206,000 1,461X10 151 10,709 Absidia ramosa (Vuillemin) Lendner x 1.32 52,300 396 10 106 8,030 (lFO 8083) o 1.31 135,200 1,032X10 127 9,695

It is clear from the foregoing table that the a-galacto- 2. A method according to c1a1m l, whereln the mold sidase activity increased by at least two times in the cu1- is a species belonging to the genus of Circinella. ture performed in the presence of 0.7 percent of citric 3. A method according to claim 1, wherein the mold acidis a species belonging to the genus of Absidia.

We claim: l. A method for the production of a-galactosidase by it 

1. A METHOD FOR THE PRODUCTION OF A-GALACTOSIDASE BY THE CULTURE OF A MOLD, BELONGING TO THE GENUS OF CIRCINELLA OR ABSIDIA WHICH COMPRISES ADDING TO A BASAL MEDIUM ABOUT 0.2 PERCENT TO ABOUT 1.0 PERCENT BASED ON SAID BASAL MEDIUM ABOUT 0.2 AN ORGANIC ACID SELECTED FROM THE GROUP CONSISTING OF CITRIC ACID LACTIC ACID, GLYCOLIC ACID, FUMARIC ACID, GLUTARIC ACID, MALIC ACID, GLACTURONIC ACID, TASTARIC ACID, SUCCINIC ACID AND PYRUVIC ACID, AND CULTURING THE MOLD IN SAID PREPARED MEDIUM UNDER CONDITIONS ENABLING THE MOLD TO PRODUCE A-GLACTOSIDASE.
 2. A method according to claim 1, wherein the mold is a species belonging to the genus of Circinella.
 3. A method according to claim 1, wherein the mold is a species belonging to the genus of Absidia. 